How to make a minimal genome for synthetic minimal cell

As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the whole-genome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.     

The synthesis of artificial genome as the basis of synthetic biology

Recent achievements in the whole-genome sequencing especially viral and bacterial ones together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes based on chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells will undoubtedly render huge influence on a deepening of knowledge on mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology.     

Whole genome amplification--applications and advances

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many--forensic science, embryonic disease diagnosis, bioterrorism genome detection, "immortalization" of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.     

Polyploid measles virus with hexameric genome length

Particles of most virus species accurately package a single genome, but there are indications that the pleomorphic particles of parainfluenza viruses incorporate multiple genomes. We characterized a stable measles virus mutant that efficiently packages at least two genomes. The first genome is recombinant and codes for a defective attachment protein with an appended domain interfering with fusion-support function. The second has one adenosine insertion in a purine run that interrupts translation of the appended domain and restores function. In that genome, a one base deletion in a different purine run abolishes polymerase synthesis, but restores hexameric genome length, thus ensuring accurate RNA encapsidation, which is necessary for efficient replication. Thus, the two genomes are complementary. The infection kinetics of this mutant indicate that packaging of multiple genomes does not negatively affect growth. We also show that polyploid particles are produced in standard infections at no expense to infectivity. Our results illustrate how the particles of parainfluenza viruses efficiently accommodate cargoes of different volume, and suggest a mechanism by which segmented genomes may have evolved.     

Mammalian chromosome banding--an expression of genome organization

Banding of metaphase chromosomes is an invaluable aid to analysing the complex genomes of vertebrates, but the biochemical basis for this phenomenon is poorly understood. Advances in molecular biology are beginning to point to features of genome organization that may play roles in chromosome banding.     

Genomorama: genome visualization and analysis

Background  

The ability to visualize genomic features and design experimental assays that can target specific regions of a genome is essential for modern biology. To assist in these tasks, we present Genomorama, a software program for interactively displaying multiple genomes and identifying potential DNA hybridization sites for assay design.     

The Ensembl genome database project

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.     

MicroRNAs: hidden in the genome

Genes for tiny RNAs have been found to be plentiful in the genomes of worms, flies, humans and probably all animals. Some of these microRNAs have been conserved through evolution, and many are expressed only at specific times or places. How they act is just beginning to be understood, but their importance to biology is likely to be great.     

Limitations of next-generation genome sequence assembly

High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics analyses and studies of genome evolution.     

Beyond the sequence: cellular organization of genome function

Genomes are more than linear sequences. In vivo they exist as elaborate physical structures, and their functional properties are strongly determined by their cellular organization. I discuss here the functional relevance of spatial and temporal genome organization at three hierarchical levels: the organization of nuclear processes, the higher-order organization of the chromatin fiber, and the spatial arrangement of genomes within the cell nucleus. Recent insights into the cell biology of genomes have overturned long-held dogmas and have led to new models for many essential cellular processes, including gene expression and genome stability.     

Dynamic bacterial genome organization

Recently completed projects of sequencing chromosomal fragments and entire chromosomes, as well as physical mapping of genomes, have opened novel inroads to the understanding of the biology of bacterial genomes. From these studies one may draw some conclusions. (i) The organization of orthologous genes on the bacterial chromosome is not conserved during evolution. (ii) The bacterial genome is more complex and also more flexible than hitherto thought. Genetic elements are sometimes part of the chromosome, while at other times they are independent elements or parts of alternative replicons (e.g. large plasmids). Such replicons, carrying essential genes, now seem to deserve the designation 'secondary chromosomes'. A study of the regulation of replication and segregation of these essential genetic elements will be of great interest.     

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.     

The Ciona intestinalis genome: when the constraints are off

The recent genome sequencing of a non-vertebrate deuterostome, the ascidian tunicate Ciona intestinalis, makes a substantial contribution to the fields of evolutionary and developmental biology.1 Tunicates have some of the smallest bilaterian genomes, embryos with relatively few cells, fixed lineages and early determination of cell fates. Initial analyses of the C. intestinalis genome indicate that it has been evolving rapidly. Comparisons with other bilaterians show that C. intestinalis has lost a number of genes, and that many genes linked together in most other bilaterians have become uncoupled. In addition, a number of independent, lineage-specific gene duplications have been detected. These new results, although interesting in themselves, will take on a deeper significance once the genomes of additional invertebrate deuterostomes (e.g. echinoderms, hemichordates and amphioxus) have been sequenced. With such a broadened database, comparative genomics can begin to ask pointed questions about the relationship between the evolution of genomes and the evolution of body plans.     

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.     

The evolving fungal genome

Fungal genomes vary considerably in size and organization. The genome of Microsporidium contains less than 3 Mb while the genomes of several Basidiomycetes and Ascomycetes greatly exceed 100 Mb. Likewise chromosome numbers and ploidy levels can differ even between closely related species. The differences in genome architecture among fungi reflect the interplay of different mutational processes as well as the population biology of the different species. Comparative genome studies have elucidated the underlying mechanisms of genome evolution in different groups of fungi and have provided insight into species-specific genomic traits. Mobile genetic elements have been instrumental in shaping the genome architecture and gene content in many fungal species. In many pathogenic fungi the mobile genetic elements even play a crucial role in rapid adaptive evolution by mediating high rates of sequence mutations, chromosomal rearrangements, and ploidy changes. But in many species mobile elements are efficiently restricted by defense mechanisms, which have evolved to suppress and regulate parasitic elements. Different rates of genome dynamic and adaptive evolution may reflect varying effective population sizes through which genetic drift and natural selection have differentially affected genome architecture in fungi over time.     

Weighted genome trees: refinements and applications

There are many ways to group completed genome sequences in hierarchical patterns (trees) reflecting relationships between their genes. Such groupings help us organize biological information and bear crucially on underlying processes of genome and organismal evolution. Genome trees make use of all comparable genes but can variously weight the contributions of these genes according to similarity, congruent patterns of similarity, or prevalence among genomes. Here we explore such possible weighting strategies, in an analysis of 142 prokaryotic and 5 eukaryotic genomes. We demonstrate that alternate weighting strategies have different advantages, and we propose that each may have its specific uses in systematic or evolutionary biology. Comparisons of results obtained with different methods can provide further clues to major events and processes in genome evolution.     

High-resolution genotyping via whole genome hybridizations to microarrays containing long oligonucleotide probes

To date, microarray-based genotyping of large, complex plant genomes has been complicated by the need to perform genome complexity reduction to obtain sufficiently strong hybridization signals. Genome complexity reduction techniques are, however, tedious and can introduce unwanted variables into genotyping assays. Here, we report a microarray-based genotyping technology for complex genomes (such as the 2.3 GB maize genome) that does not require genome complexity reduction prior to hybridization. Approximately 200,000 long oligonucleotide probes were identified as being polymorphic between the inbred parents of a mapping population and used to genotype two recombinant inbred lines. While multiple hybridization replicates provided ～97% accuracy, even a single replicate provided ～95% accuracy. Genotyping accuracy was further increased to >99% by utilizing information from adjacent probes. This microarray-based method provides a simple, high-density genotyping approach for large, complex genomes.